ERCC1 Overexpression Increases Radioresistance in Colorectal Cancer Cells

Simple Summary The 20–30% of locally advanced rectal cancer patients undergoing preoperative concurrent chemoradiotherapy had no expected efficacy, and ERCC1 overexpression was found in these tumor tissue patients. In the interest of confirming and adding details to our understanding of that correlation, The Tet-on gene expression system was used to examine ERCC1 functionality and stability. Our data from regulatable HCT116-Tet-on and COLO205-Tet-on cell lines verified the increased radioresistance in colorectal cancer cells that are associated with ERCC1 overexpression, and they confirmed a high correlation between ERCC1 levels and radiotherapeutic efficiency. Furthermore, overexpression of ERCC1 also increases cell migration under radiation exposure. Additional data from ERCC1 expression regulation in vivo confirmed that the overexpression of increased cancer radiation resistance suggests that ERCC1 expression plays a key role. Abstract Preoperative concurrent chemoradiotherapy (CCRT) is a standard treatment for locally advanced rectal cancer patients, but 20–30% do not benefit from the desired therapeutic effects. Previous reports indicate that high levels of ERCC1 reduce the effectiveness of cisplatin-based CCRT; however, it remains unclear as to whether ERCC1 overexpression increases radiation resistance. To clarify the correlation between ERCC1 levels and radiation (RT) resistance, we established two cell lines (HCT116-Tet-on and COLO205-Tet-on), induced them to overexpress ERCC1, detected cell survival following exposure to radiation, established HCT116-Tet-on and COLO205-Tet-on heterotopic cancer animal models, and detected tumor volume following exposure to radiation. We found that ERCC1 overexpression increased radiation resistance. After regulating ERCC1 levels and radiation exposure to verify the correlation, we noted that increased radiation resistance was dependent on ERCC1 upregulation in both cell lines. For further verification, we exposed HCT116-Tet-on and COLO205-Tet-on heterotopic cancer animal models to radiation and observed that ERCC1 overexpression increased colorectal cancer tumor radioresistance in both. Combined, our results suggest that ERCC1 overexpression may serve as a suitable CCRT prognostic marker for colorectal cancer patients.

Introduction
A total of 104,270 new colon cancer cases, 45,230 new rectal cancer cases, and 52,980 associated deaths were reported in 2021 alone in the United States [1]. Colorectal cancer was identified as the third most prevalent cancer type in the US for that year [2], with local recurrence rates of resected patients with stage II-III rectal cancer ranging from 15% to 65%. Local recurrence rates for stage III patients following total mesorectal excision (TME) surgery have been reported as ranging from 20% to 30% [3][4][5]. Limiting postoperative recurrence is a major treatment priority for colorectal cancer patients.
To improve cure rates and reduce local recurrence, the National Comprehensive Cancer Network recommends preoperative concurrent chemoradiotherapy (CCRT) as a priority treatment for locally advanced rectal cancers [6,7]. Among patients receiving preoperative radiotherapy, one research team has reported a 61% reduction in relative local recurrence risk (95% CI: 0.27-0.58, p < 0.0001), with a 6.2% absolute difference at three years (95% CI: 5.3-7.1) and a 24% relative improvement in disease-free survival (HR = 0.76, 95% CI: 0.62-0.94, p = 0.013) [8]. According to this study, among others, CCRT treatment of colorectal cancer is now considered indispensable; however, some patients exhibit a poor response to CCRT, thus increasing the potential for tumor progression and additional resection surgery. Three specific issues that must be addressed are (i) patients who do not respond well to CCRT often cannot obtain accurate preoperative pathologic staging for early rectal cancer (T1T2N0), thus increasing the potential for staging overestimates during preoperative clinical imaging, which makes treatment modelling decisions more difficult [9,10]; (ii) additional surgery entails increased difficulties, especially for shortterm hypofractionated radiotherapy-more specifically, surgeons must deal with inflamed tumors and the surrounding tissue being represented as congestion or edema, both of which suggest intraoperative bleeding [11]; and (iii) after long-term preoperative radiotherapy, tissue becomes fibrotic, and tumors adhere to surrounding structures, thus making surgical separation more difficult and triggering postoperative complications such as anastomotic leakage (LAR surgery) and perineal wound dehiscence (APR surgery) [3].
Finally, and most importantly, patients who are insensitive to radiotherapy, chemotherapy, and neoadjuvant chemoradiotherapy may experience delayed opportunities for resection surgery, thus allowing for greater tumor development [12]. In short, the tumor edge blur ring in CCRT-insensitive patients stands as a major challenge against successful resection surgery due to inaccuracies in measuring CCRT efficacy potential [11].
We previously reported finding a high correlation between ERCC1 gene overexpression and radiation response in colorectal cancer patients [13]. In the interest of confirming and adding details to our understanding of that correlation, we used controlled gene expression tetracycline technology (Tet-on system) to establish stable ERCC1 expression in two colorectal cell lines to verify the effects of different ERCC1 concentrations on radiation resistance. We then used an ectopic colorectal cancer animal model to investigate the effects, if any, of ERCC1 overexpression on colorectal cancer radiation sensitivity.

Plasmid Construction
The Tet-on ERCC1 system was generated by using a Pas4.1w. Ppuro-aOn cassette (RNAi coil, Academia Sinica, Taipei, Taiwan) and inserting the nucleotide sequence of the ERCC1 isoform1 (also known as: 202, NCBI identifier: P07992-1) which was constructed by PCR amplification using the following primers: CTAGCTAGCAAGCTTGC-CACCATGGACCCTGGGAAGGAC, which has a NheI site, and CCGGCGCGCCGTT-TAAACATCGATTCACAGATCCTCTTCTGAGATGAGTTTTTGTTCGGG, which has myc Tag and PmeI site.

Cell Lines and Generation of Tet-on Ercc1 Stable Cell Lines
HCT116 and COLO 205 colon cancer cell lines (American Type Culture Collection) were cultured in DMEM (Sigma-Aldrich, St. Louis, MI, USA), supplemented with 10% bovine calf serum, 100 units/mL penicillin, and 100 µg/mL streptomycin at 37 • C in an atmosphere of 5% CO 2 . Tet-on ERCC1 stable cell lines were established via the transfection of a recombinant lentivirus that consisted of a package plasmid, pCMV∆R8.91 (RNAi coil, Academia Sinica, Taipei, Taiwan), pMD.G (RNAi coil, Academia Sinica, Taiwan), and a target plasmid, the Tet-On ERCC1 system, respectively. After selection by 1, 3, and 5 µg/mL Puromycin dihydrochloride (P8833, Sigma-Aldrich), the selection medium was replaced every 2 days for one week.

Irradiation Exposes, CELL viability Assay, and Colony Formation Assay
Cells were plated in a 96-well plate (2000/well) and irradiated overnight with 2 Gy per time linear accelerator (Varian Clinac iX, Department of Radiation Oncology, Kaohsiung Medical University Hospital), before a bolus (1.5-cm-thick) was placed on the top and bottom of cell culture plates. Cell viability was measured using an ATPlite kit (510-17281, Blossom Biotechnologies, Taipei, Taiwan), and the luminesce value was measured using a multimode plate reader (VICTORTM X2, PerkinElmer, Waltham, MA, USA). In the colony formation assay, 2000 cells per well were seeded on a 6-well plate (2000/well) and incubated in an incubator for 14 days after irradiation, using the radiation protocol described above. Using a 1% crystal violet (C0775-25G, MERCK)/methanol (67-56-1, MERCK) buffer, the cells were stained for 10 min, washed with ddH 2 O, dried, and the colonies were counted.

Wound Healing Assay
Cells were seeded in 6-well plates at 80% of confluence in a growth medium. A 0.4-mm wide wound was performed after cells were attached to the plate using a 10 µL tip. Wound images were taken during 48 h using Olympus microscope (Olympus Microsystems). Images were quantified with ImageJ software (image J (ImageJ bundled with 64-bit Java 1.8.0_172, Wayne Rasband, Bethesda, Rockville, MD, USA, https://imagej.net/imagejwiki-static/Downloads (accessed on 28 June 2022)). The cell-free area was normalized on day 0. The quantification of the cell-free area (%) is as follows: cell-free area (%) = (cell-free area of Day 0, Day 1, or Day 2, mm 3 )/(the cell-free area of Day 0, mm 3 ) × 100%.

Xenograft Animal Model and Radiation Exposure Schedule
Specific pathogen-free male BALB/c nude mice were obtained from the National Laboratory Animal Center, Taipei, Taiwan. All animal experiments were conducted in accordance with institutional guidelines and approved by the Animal Care and Use Committee of the Kaohsiung Medical University. Tet-on ERCC1, HCT116, and Tet-on ERCC1 COLO 205 cells were subcutaneously injected into 2 × 10 6 cells in nude mice, respectively. When tumors reached a size of approximately 50 mm 2 , mice were divided into three groups: buffer alone (control), and doxycycline at 10 or 50 mg/kg/mouse by intraperitoneal injection. Radiation was performed with multiple doses of 2 Gy per fraction (cumulative radiation dose was 6 Gy). There were four mice in each group. After sacrificing the mice, the tumor mass was removed and analyzed for ERCC1 expression. In the in vivo experiment, HCT116 and COLO 205 tumor-bearing mice underwent irradiation. Mice were divided into four groups: Group 1 (-ERCC1-RT), in which mice were not treated with either a doxycycline injection or radiation therapy; Group 2 (+ERCC1-RT), in which mice were injected with doxycycline, but received no radiation; Group 3 (-ERCC1+RT), in which mice were not injected with doxycycline, but were treated with radiation; and Group 4 (+ERCC1+RT), in which mice were injected with doxycycline and received radiation. The tumor size was measured by using digital calipers (VWR International, Radnor, PA, USA), and the tumor volume was determined using the following formula: tumor volume [mm 3 ] = (length [mm]) × (width [mm]) 2 × 0.52. Tumor weight was measured using an electronic scale.

Statistical Analysis
Statistical significance was calculated using GraphPad Prism 6.0 with Student's t-test and multiple t-tests. Data were considered statistically different at p < 0.05.

Establishing a Doxycycline-Inducible Gene Expression System to Regulate ERCC1 and in HCT116 and COLO205
To select colorectal cancer cells with low ERCC1 expression, we analyzed the expression of ERCC1 mRNA and the protein in HEK293, HCT116, SW620, COLO205, and SW480 cell lines (Supplementary Figure S1). The results showed the expression of ERCC1 in HCT116 and COLO205 to be lower than other colorectal cancer cell lines; therefore, we used HCT116 and COLO205 for the establishment of Tet on ERCC1 stable cell lines. A Tet-on ercc1-myc Tag system ( Figure 1A) was used to construct HCT116 and COLO205 colorectal cell lines to examine ERCC1 expression. We compared untreated cells with cells treated with doxycycline for 24 h to determine the successful establishment of HCT116-Tet-on-ERCC1 and COLO205-Tet-on-ERCC1 cell lines. Both anti-MYC and antiERCC1 antibody detection data indicate that compared with a mock wild-type cell line and the nondoxycycline (−) group, the doxycycline group (+) expressed high levels of ERCC1 in both cell lines ( Figure 1B,C)-thus, it is evident that HCT116 and COLO 205 were successfully established.
Tet-on-ERCC1 and COLO205-Tet-on-ERCC1 cell lines. Both anti-MYC and antiERCC1 antibody detection data indicate that compared with a mock wild-type cell line and the nondoxycycline (-) group, the doxycycline group (+) expressed high levels of ERCC1 in both cell lines ( Figure 1B,C)-thus, it is evident that HCT116 and COLO 205 were successfully established.

ERCC1 Overexpression Confers Radiation Resistance in-Tet-on-ERCC1 Cell Lines
Cells were divided into three groups: WT (wild type), a Tet-on group without doxycycline treatment Tet-on-ERCC1 (-), and a Tet-on-ERCC1 (+) group. These groups were treated with 2 μg/mL doxycycline for 24 h. Cell viability was analyzed using an ATPlite kit 72 h after radiation exposure at 6-16 Gy. Half-maximal inhibitory concentrations for the HCT116 cell line were 11.17 ± 0.13 Gy for mock wt cells, 9.75 ± 0.06 Gy for Tet-on cells, and 14.12 ± 0.83 Gy for Tet-on + Dox cells ( Figure 2A). The respective concentrations for the COLO 205 cell line were 10.29 ± 0.11 Gy, 10.43 ± 0.25 Gy, and 11.65 ± 0.25 Gy ( Figure 2B). These results confirm radiation resistance in HCT116-Tet-on-ERCC1 and COLO205-Tet-on-ERCC1 cells conferred by ERCC1 overexpression. To further confirm that the increased radiation tolerance by overexpression of ERCC1 is due to cell proliferation, we used the colony formation for analysis, and HCT116-Tet-on-ERCC1 and COLO205-Tet-on-ERCC1 cells were treated. Then, 0, 0.5, 1.0, 2 μg/mL doxycycline was given with 8 Gy of radiation, and they were left to react for 14 days; the number of colonies formed in each group was subsequently analyzed (%). The results suggested that the number of cell populations in the overexpressed ERCC1 group is greater than the different radiation doses in the HCT116-Tet-on-ERCC1 ( Figure 2C,E) and COLO205-Tet-on-ERCC1 cell groups ( Figure 2D,F). This phenomenon indicates that the radioresistance of overexpressed ERCC1 reduces the severity of restrictions placed on radiation therapy when treating cell proliferation.

ERCC1 Overexpression Confers Radiation Resistance in-Tet-on-ERCC1 Cell Lines
Cells were divided into three groups: WT (wild type), a Tet-on group without doxycycline treatment Tet-on-ERCC1 (−), and a Tet-on-ERCC1 (+) group. These groups were treated with 2 µg/mL doxycycline for 24 h. Cell viability was analyzed using an ATPlite kit 72 h after radiation exposure at 6-16 Gy. Half-maximal inhibitory concentrations for the HCT116 cell line were 11.17 ± 0.13 Gy for mock wt cells, 9.75 ± 0.06 Gy for Tet-on cells, and 14.12 ± 0.83 Gy for Tet-on + Dox cells ( Figure 2A). The respective concentrations for the COLO 205 cell line were 10.29 ± 0.11 Gy, 10.43 ± 0.25 Gy, and 11.65 ± 0.25 Gy ( Figure 2B). These results confirm radiation resistance in HCT116-Tet-on-ERCC1 and COLO205-Tet-on-ERCC1 cells conferred by ERCC1 overexpression. To further confirm that the increased radiation tolerance by overexpression of ERCC1 is due to cell proliferation, we used the colony formation for analysis, and HCT116-Tet-on-ERCC1 and COLO205-Tet-on-ERCC1 cells were treated. Then, 0, 0.5, 1.0, 2 µg/mL doxycycline was given with 8 Gy of radiation, and they were left to react for 14 days; the number of colonies formed in each group was subsequently analyzed (%). The results suggested that the number of cell populations in the overexpressed ERCC1 group is greater than the different radiation doses in the HCT116-Tet-on-ERCC1 ( Figure 2C,E) and COLO205-Tet-on-ERCC1 cell groups ( Figure 2D,F). This phenomenon indicates that the radioresistance of overexpressed ERCC1 reduces the severity of restrictions placed on radiation therapy when treating cell proliferation.
tibody detection data indicate that compared with a mock wild-type cell line and the nondoxycycline (-) group, the doxycycline group (+) expressed high levels of ERCC1 in both cell lines ( Figure 1B,C)-thus, it is evident that HCT116 and COLO 205 were successfully established.

ERCC1 Overexpression Confers Radiation Resistance in-Tet-on-ERCC1 Cell Lines
Cells were divided into three groups: WT (wild type), a Tet-on group without doxycycline treatment Tet-on-ERCC1 (-), and a Tet-on-ERCC1 (+) group. These groups were treated with 2 μg/mL doxycycline for 24 h. Cell viability was analyzed using an ATPlite kit 72 h after radiation exposure at 6-16 Gy. Half-maximal inhibitory concentrations for the HCT116 cell line were 11.17 ± 0.13 Gy for mock wt cells, 9.75 ± 0.06 Gy for Tet-on cells, and 14.12 ± 0.83 Gy for Tet-on + Dox cells (Figure 2A). The respective concentrations for the COLO 205 cell line were 10.29 ± 0.11 Gy, 10.43 ± 0.25 Gy, and 11.65 ± 0.25 Gy ( Figure 2B). These results confirm radiation resistance in HCT116-Tet-on-ERCC1 and COLO205-Tet-on-ERCC1 cells conferred by ERCC1 overexpression. To further confirm that the increased radiation tolerance by overexpression of ERCC1 is due to cell proliferation, we used the colony formation for analysis, and HCT116-Tet-on-ERCC1 and COLO205-Tet-on-ERCC1 cells were treated. Then, 0, 0.5, 1.0, 2 μg/mL doxycycline was given with 8 Gy of radiation, and they were left to react for 14 days; the number of colonies formed in each group was subsequently analyzed (%). The results suggested that the number of cell populations in the overexpressed ERCC1 group is greater than the different radiation doses in the HCT116-Tet-on-ERCC1 ( Figure 2C,E) and COLO205-Tet-on-ERCC1 cell groups ( Figure 2D,F). This phenomenon indicates that the radioresistance of overexpressed ERCC1 reduces the severity of restrictions placed on radiation therapy when treating cell proliferation.

Overexpression of ERCC1 Enhances the HCT116-Tet-on-ERCC1 and COLO205-Tet-on-ERCC1 Migration after Radiation Exposure
To clarify whether the overexpression of ERCC1 and radiation exposure following ERCC1 overexpression could affect the proliferation and migration ability of colorectal cancer cells, HCT116-Tet-on-ERCC1 and COLO205-Tet-on-ERCC1 were divided into no expression ERCC1-, overexpression ERCC1 (treated with 2 μg/mL), no expression ERCC1 combined with radiation exposure, and overexpression ERCC1 combined with radiation exposure; ATP Lite analysis was performed at 24 h and 48 h. The results suggest that compared with the non-ERCC1 expression group, the luminescence intensity of overexpressed ERCC1 is not higher than that of the non-ERCC1 expression group, indicating that the presence or absence of overexpressed ERCC1 has no correlation with colorectal cancer cells. Furthermore, to analyze whether the overexpression of ERCC1 has an effect on cell proliferation under radiation irradiation, the results show that under radiation irradiation, the overexpression of ERCC1 can increase cell proliferation in HCT116 Tet on ( Figure 4A) and COLO205 Tet on ( Figure 4B). Furthermore, we analyzed whether the overexpression of ERCC1 in this environment could increase the migration ability of HCT116 Tet on and COLO205 Tet on. The results showed that the overexpression of ERCC1 in both HCT116 Tet on ( Figure 4C) and COLO205 Tet on ( Figure 4D) increased their migration ability when exposed to radiation ( Figure 4E,F). This phenomenon indicates that the overexpression of ERCC1 could increase the proliferation and migration

Overexpression of ERCC1 Enhances the HCT116-Tet-on-ERCC1 and COLO205-Tet-on-ERCC1 Migration after Radiation Exposure
To clarify whether the overexpression of ERCC1 and radiation exposure following ERCC1 overexpression could affect the proliferation and migration ability of colorectal cancer cells, HCT116-Tet-on-ERCC1 and COLO205-Tet-on-ERCC1 were divided into no expression ERCC1-, overexpression ERCC1 (treated with 2 µg/mL), no expression ERCC1 combined with radiation exposure, and overexpression ERCC1 combined with radiation exposure; ATP Lite analysis was performed at 24 h and 48 h. The results suggest that compared with the non-ERCC1 expression group, the luminescence intensity of overexpressed ERCC1 is not higher than that of the non-ERCC1 expression group, indicating that the presence or absence of overexpressed ERCC1 has no correlation with colorectal cancer cells. Furthermore, to analyze whether the overexpression of ERCC1 has an effect on cell proliferation under radiation irradiation, the results show that under radiation irradiation, the overexpression of ERCC1 can increase cell proliferation in HCT116 Tet on ( Figure 4A) and COLO205 Tet on ( Figure 4B). Furthermore, we analyzed whether the overexpression of ERCC1 in this environment could increase the migration ability of HCT116 Tet on and COLO205 Tet on. The results showed that the overexpression of ERCC1 in both HCT116 Tet on ( Figure 4C) and COLO205 Tet on ( Figure 4D) increased their migration ability when exposed to radiation ( Figure 4E,F). This phenomenon indicates that the overexpression of ERCC1 could increase the proliferation and migration ability of colorectal cancer cells in a radiation-irradiated environment. We evaluated the association of the ERCC1 level with/without radiation exposure between the markers of MET and EMT in rectal cancer cells. HCT116-Tet-on-ERCC1 and COLO205-Tet-on-ERCC1 were treated with 0 µg/mL or 2 µg/mL Dox to induce the overexpression of ERCC1. After 24 h, the cells were exposed with/without the 8 Gy radiation. The results suggested that the ERCC1 level, and the level of radiation that the cells were exposed to, were not key in terms of affecting tumor generation (Supplementary Figure S2). This is because the overexpression of ERCC1, and the exposure to radiation, did not alter the levels of MET and EMT markers in CRC cell lines compared with controls; this is perhaps due to the fact that ERCC1 is a downstream factor of DNA repair and does not affect tumorigenesis. Based on the above data and the aforementioned arguments of this study, the overexpression of ERCC1 may only regulate resistance to chemotherapy or radiotherapy without affecting tumorigenesis. ability of colorectal cancer cells in a radiation-irradiated environment. We evaluated the association of the ERCC1 level with/without radiation exposure between the markers of MET and EMT in rectal cancer cells. HCT116-Tet-on-ERCC1 and COLO205-Tet-on-ERCC1 were treated with 0 μg/mL or 2 μg/mL Dox to induce the overexpression of ERCC1. After 24 h, the cells were exposed with/without the 8 Gy radiation. The results suggested that the ERCC1 level, and the level of radiation that the cells were exposed to, were not key in terms of affecting tumor generation (Supplementary Figure S2). This is because the overexpression of ERCC1, and the exposure to radiation, did not alter the levels of MET and EMT markers in CRC cell lines compared with controls; this is perhaps due to the fact that ERCC1 is a downstream factor of DNA repair and does not affect tumorigenesis. Based on the above data and the aforementioned arguments of this study, the overexpression of ERCC1 may only regulate resistance to chemotherapy or radiotherapy without affecting tumorigenesis.

Establishing an Animal Model for Precise ERCC1 Regulation
HCT116-Tet-on and COLO205-Tet-on heterotopic cancer animal models were established to verify the induction of ERCC1 expression in tumors via in vivo injection of doxycycline. Tumors were subcutaneously injected into mice to generate HCT116-Tet-on and COLO205-Tet-on tumor-bearing mice. Doxycycline (10 mg/kg or 50 mg/kg) was IP injected when the tumor volume reached 50 mm 3 . Tumors were collected 24 h postinjection and examined using Western blotting to detect ERCC1 expression. Results show that doxycycline treatment at 50 mg/kg was capable of regulating ERCC1 overexpression in vivo ( Figure 5A,B), thus confirming the successful establishment of an animal model for precise ectopic cancer ERCC1 regulation.

Establishing an Animal Model for Precise ERCC1 Regulation
HCT116-Tet-on and COLO205-Tet-on heterotopic cancer animal models were established to verify the induction of ERCC1 expression in tumors via in vivo injection of doxycycline. Tumors were subcutaneously injected into mice to generate HCT116-Tet-on and COLO205-Tet-on tumor-bearing mice. Doxycycline (10 mg/kg or 50 mg/kg) was IP injected when the tumor volume reached 50 mm 3 . Tumors were collected 24 h post-injection and examined using Western blotting to detect ERCC1 expression. Results show that doxycycline treatment at 50 mg/kg was capable of regulating ERCC1 overexpression in vivo ( Figure 5A,B), thus confirming the successful establishment of an animal model for precise ectopic cancer ERCC1 regulation.

ERCC1 Overexpression Induced Radiation Resistance and Reduced Radiationtherapeutic Efficiency
HCT116-Tet-on or COLO205-Tet-on tumors were subcutaneously injected into nude mice to generate HCT116-Tet-on-ERCC1 or COLO205-Tet-on-ERCC1 tumor-bearing mice. Mice were IP-treated with 50 mg/kg doxycycline or PBS and exposed to 6 Gy radiation in three fractions over 7 days. Tumors were measured every 3 days for 33 days ( Figure  6A,B), characterized ( Figure 6C,D), and then weighed ( Figure 6E,F) at the end of the experiment. Our results indicate that there was a larger tumor volume and higher tumor weight in the +Dox +RT group compared with the +RT group on day 33. These results confirm the increased tumor resistance to radiation due to ERCC1 overexpression.

ERCC1 Overexpression Induced Radiation Resistance and Reduced Radiationtherapeutic Efficiency
HCT116-Tet-on or COLO205-Tet-on tumors were subcutaneously injected into nude mice to generate HCT116-Tet-on-ERCC1 or COLO205-Tet-on-ERCC1 tumor-bearing mice. Mice were IP-treated with 50 mg/kg doxycycline or PBS and exposed to 6 Gy radiation in three fractions over 7 days. Tumors were measured every 3 days for 33 days ( Figure 6A,B), characterized ( Figure 6C,D), and then weighed ( Figure 6E,F) at the end of the experiment. Our results indicate that there was a larger tumor volume and higher tumor weight in the +Dox +RT group compared with the +RT group on day 33. These results confirm the increased tumor resistance to radiation due to ERCC1 overexpression. Figure 6. ERCC1 overexpression reduced RT-therapeutic efficiency. A xenograft model was divided into four groups: control, +Dox (treated with doxycycline), +RT (exposed to radiation), and +Dox +RT (treated with doxycycline and exposed to radiation). Tumor volumes were measured between days 0 and 33 in (A) HCT116-Tet-on ERCC1 and (B) COLO205-Tet-on-ERCC1 cells. (C) HCT116 (D) and COLO 205 tumors were collected and weighed on day 33. Respective weight data are shown as subfigures (E,F) (p < 0.05 *).

Discussion
The Tet-on gene expression system was used to examine ERCC1 functionality and stability. Our data from regulatable HCT116-Tet-on and COLO205-Tet-on cell lines verified the increased radioresistance in colorectal cancer cells that are associated with ERCC1 overexpression, and they confirmed a high correlation between ERCC1 levels and radiotherapeutic efficiency. Additional data from ERCC1 expression regulation in vivo confirmed that the overexpression of increased cancer radiation resistance suggests that ERCC1 expression plays a key role.
We found that ERCC1 levels affected chemotherapy and radiation therapy efficacy via DNA repair. Multiple CCRT-associated resistance factor studies have reported high correlations between ERCC1 expression and CCRT clinical efficacy. Xu et al. reported respective CCRT response rates of 76.9% and 56.6% for ERCC1 − and ERCC1 + patients with regionally moderate advanced nasopharyngeal carcinomas [14,15], and Jun et al. reported a significantly higher three-year overall survival rate in patients with low ERCC1 expression (91.7; 95% CI, 76.0-100.0%) compared with high ERCC1 expression patients (45.5; 95% CI, 23.9-67.1%; p = 0.013). Combined, these data suggest that ERCC1 overexpression Figure 6. ERCC1 overexpression reduced RT-therapeutic efficiency. A xenograft model was divided into four groups: control, +Dox (treated with doxycycline), +RT (exposed to radiation), and +Dox +RT (treated with doxycycline and exposed to radiation). Tumor volumes were measured between days 0 and 33 in (A) HCT116-Tet-on ERCC1 and (B) COLO205-Tet-on-ERCC1 cells. (C) HCT116 (D) and COLO 205 tumors were collected and weighed on day 33. Respective weight data are shown as subfigures (E,F) (p < 0.05 *).

Discussion
The Tet-on gene expression system was used to examine ERCC1 functionality and stability. Our data from regulatable HCT116-Tet-on and COLO205-Tet-on cell lines verified the increased radioresistance in colorectal cancer cells that are associated with ERCC1 overexpression, and they confirmed a high correlation between ERCC1 levels and radiotherapeutic efficiency. Additional data from ERCC1 expression regulation in vivo confirmed that the overexpression of increased cancer radiation resistance suggests that ERCC1 expression plays a key role.
We found that ERCC1 levels affected chemotherapy and radiation therapy efficacy via DNA repair. Multiple CCRT-associated resistance factor studies have reported high correlations between ERCC1 expression and CCRT clinical efficacy. Xu et al. reported respective CCRT response rates of 76.9% and 56.6% for ERCC1 − and ERCC1 + patients with regionally moderate advanced nasopharyngeal carcinomas [14,15], and Jun et al. reported a significantly higher three-year overall survival rate in patients with low ERCC1 expression (91.7; 95% CI, 76.0-100.0%) compared with high ERCC1 expression patients (45.5; 95% CI, 23.9-67.1%; p = 0.013). Combined, these data suggest that ERCC1 overexpression leads to CCRT resistance. Other research teams have reported high correlations between low ERCC1 expression and in vitro sensitivity to cisplatin in malignant cervical cancer cell lines [16,17], testicular cancer [18], and malignant effusions collected from non-small cell lung cancer patients [19,20]. One retrospective analysis showed a relationship between increased ERCC1 mRNA or protein expression levels and resistance to cisplatin-based chemotherapy in many types of advanced tumors, including non-small cell lung cancer (NSCLC) [21,22], esophageal cancer [23], and colon cancer [24][25][26][27]. In addition, we analyzed the effect of ERCC1 on colorectal cancer patients in the GEPIA database. According to the GEPIA database analysis, colon adenocarcinoma cell lines showed a higher expression of ercc1 mRNA compared with normal cells. Furthermore, we analyzed the correlation between the expression of ercc1 mRNA with the overall survival rate and disease-free survival rate in patients with colon adenocarcinoma. The results showed that the overexpression of ERCC1 did not affect the overall survival rate and disease-free survival rate in patients with rectal cancer; however, our previous study found that the overexpression of ERCC1 in colorectal cancer patients undergoing concurrent chemoradiotherapy has a poor prognosis [28]. Initially, we considered that the overexpression of ERCC1 would only affect staged outcomes of chemotherapy and radiotherapy in patients with rectal cancer, but not the overall survival rate.
The possible mechanism of the expression of ERCC1 on radiotherapy sensitivity could be due to the fact that radiation could cause double-strand breaks and single-strand breaks in cellular DNA through free radicals to limit the progression of cancer [29,30]. Many studies reported that ERCC1 plays an important role in the repair of DNA double-strand breaks and DNA single-strand breaks [31,32]. In double-strand breaks, the ERCC1/XPF complex would recognize and cleave AT-rich DNA sequences to facilitate subsequent DNA replication or conjugation. Furthermore, ERCC1 would stabilize DNA repair and accelerate the process of DSB repair [33,34]. In the process of single-stranded DNA repair, ERCC1-XPF cuts the damaged strand 5 to remove the damaged base, creating 3 -OH in order to prime DNA synthesis to complete DNA repair and reduce cancer cell apoptosis [35,36]. Based on these mechanisms, we hypothesized that the overexpression of ERCC1 would increase the ability of DNA to repair, and that it would help increase DNA's tolerance to radiotherapy.
The Tet-on gene expression system offers real-time and precise regulation of cell cycle, inflammatory, DNA repair, and other factors requiring short-term regulation. To study cell cycle factors, Kok et al. regulated Tet-on Cyclin E1 and Tet-a on Cdc25A expression in RPE1 cells [37]. When addressing DNA repair, Mladenov et al. used the Tet-on system to determine the role of the ATR promoter in the repair process [38]. Note that ERCC1 is a DNA damage-induced protein involved in nucleotide excision repair, DNA interstrand crosslink repair [39], and DNA double-strand break repair [40,41]. Certain homologous recombination repair and non-homologous end-joining pathways depend on the ERCC1-XPF function [42][43][44]. Tet-on is being increasingly used to study DNA repairrelated genes such as ERCC1 due to its precision when regulating expression. Since DNA repair-related proteins are only induced by DNA damage, rather than continuously expressed in cells, Tet-on is believed to provide accurate simulations of natural processes in vivo. According to our results, the precise control of ERCC1 expression was doxycycline concentrationdependent both in vitro ( Figure 3A,B) and in vivo ( Figure 5A,B), thus confirming the appropriateness of using Tet-on to analyze the ERCC1 system. To clarify whether the overexpression of ERCC1 affects other DNA repair pathways, we analyzed the expression of XPF, ATM, and ATR in ERCC1-overexpressing CRC cells. The results indicate that the overexpression of ERCC1 would only have increased the level of XPF. In this isoform, XPF is a DNA endonuclease, and ERCC1 is an auxiliary protein that maintains the function of XPF (Supplementary Figure S3); therefore, we may speculate that the overexpression of ERCC1 could increase the stability of XPF and reduce the degradation of XPF.
Biomarkers are commonly used for colorectal cancer diagnosis, prognosis, and treatment purposes. Kirsten rat sarcoma virus (KRAS) and N-BRAF mutations help physicians determine the potential for anti-EGFR therapy in terms of increasing EGFR antibody therapy efficacy [45]. CA 19-9 and carcinoembryonic antigens are known to improve early colorectal cancer detection and recurrence following colorectal cancer surgery [46,47]. Microsatellite instability testing is also widely used for chemotherapy prognostic assessment purposes [48]; however, there are no biomarkers that can be used as CCRT prognostic predictors, even though it is considered an important therapeutic regimen.
Data from clinical staining studies have shown that colorectal cancer patients with a poor prognosis following the combination of FOLFOX and radiation exposure often exhibit high levels of ERCC1 overexpression [49]. To confirm the effects of ERCC1 expression on CCRT efficacy, we induced ERCC1 overexpression in colorectal cancer cells and in an ectopic carcinoma animal model prior to radiation exposure, and we found evidence that ERCC1 overexpression triggered resistance to radiotherapy (Figures 2-5). According to recent reports, microsatellite instability can be used as an indicator of FOLFOX clinical efficacy, thus suggesting that DNA repair robustness can affect colorectal cancer responsiveness to chemotherapy and radiotherapy. There is currently interest in testing an ERCC1 inhibitor as a CCRT sensitizer.

Conclusions
Our in vitro and in vivo results indicate that ERCC1 overexpression triggered colorectal cancer radioresistance; therefore, we believe that ERCC1 levels may affect chemotherapy and radiation therapy efficacy via DNA repair. Further research is required to confirm ERCC1 expression detection as an appropriate predictor of CCRT treatment efficacy, and to test the possibility of combining ERCC1 inhibitors to improve CCRT efficacy for colorectal cancer patients. Institutional Review Board Statement: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.

Data Availability Statement:
The data used to support the findings of this study are included in the article.

Conflicts of Interest:
All authors declare that they have no conflict of interest. There are no financial relationships or otherwise that might lead to a conflict of interest. No published data or illustrations are reproduced in the paper.


Aqueous extract of Artemisia capillaris improves non-alcoholic fatty liver and obesity in mice induced by high-fat diet

Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases and is a nutritional metabolic disease. Artemisia capillaris (AC) is the above-ground dried part of Artemisia capillaris Thunb. or Artemisia scoparia Waldst. et Kit., a natural medicinal plant with pharmacological effects of heat-clearing and biliary-promoting. In order to evaluate the therapeutic effect of Artemisia capillaris on NAFLD and obesity, experiments were conducted using aqueous extracts of Artemisia capillaris (WAC) to intervene in NAFLD models in vivo and in vitro. In vivo experiments were performed using HFD-fed (high fat diet) C57BL/6 mice to induce NAFLD model, and in vitro experiments were performed using oleic acid to induce HepG2 cells to construct NAFLD cell model. H.E. staining and oil red O staining of liver tissue were used to observe hepatocytes. Blood biochemistry analyzer was used to detect serum lipid levels in mice. The drug targets and mechanism of action of AC to improve NAFLD were investigated by western blotting, qRT-PCR and immunofluorescence. The results showed that C57BL/6 mice fed HFD continuously for 16 weeks met the criteria for NAFLD in terms of lipid index and hepatocyte fat accumulation. WAC was able to reverse the elevation of serum lipid levels induced by high-fat diet in mice. WAC promoted the phosphorylation levels of PI3K/AKT and AMPK in liver and HepG2 cells of NAFLD mice, inhibited SREBP-1c expression, reduced TG and lipogenesis, and decreased lipid accumulation. In summary, WAC extract activates PI3K/AKT pathway, reduces SREBP-1c protein expression by promoting AMPK phosphorylation, and decreases fatty acid synthesis and TG content in hepatocytes. AC can be used as a potential health herb to improve NAFLD and obesity.

Introduction
Non-alcoholic fatty liver disease (NAFLD) is a clinicopathologic syndrome of hepatic steatosis. It is characterized by excessive accumulation of lipids, mainly triglycerides (TG), in hepatocytes. However, NAFLD does not include a history of excessive alcohol consumption and definite liver damage factors (Cohen et al., 2011;Chalasani et al., 2018). NAFLD is a chronic nutritional metabolic disease that can develop into hepatitis, cirrhosis, and even liver cancer (Diehl and Day, 2017;Huang et al., 2021;Ratziu et al., 2022). Studies have shown that the incidence of NAFLD has shown a high development worldwide (Lonardo et al., 2016). It is closely associated with other metabolic diseases such as obesity, type 2 diabetes, dyslipidemia and hypertension, so NAFLD has become a global health problem of concern (Chalasani et al., 2018). Currently, clinical treatment is mostly carried out with lipid-lowering drugs, antioxidants and insulin sensitizers, but long-term use of these drugs produces toxic side effects. Natural medicines as alternative therapies have their unique advantages in the treatment of NAFLD, and there are some herbal medicines that have potential applications in the treatment of NAFLD.
The occurrence of NAFLD is closely related to insulin resistance (IR). When IR occurs in the body, in order to meet the body's energy supply needs, fat becomes the main mode of energy supply. After the increased breakdown of peripheral adipose tissue in the body, the uptake of free fatty acids by hepatocytes increases and they are deposited in the form of TG in the cells, and eventually NAFLD occurs. The phosphatidylinositol-3-kinase (PI3K) signaling pathway (PI3K/ AKT) is the classical hepatic insulin signaling pathway (Fukuda et al., 2016). Activation of the PI3K/AKT transduction pathway improves IR and alleviates NAFLD. AMP-activated protein kinase (AMPK) is an important factor in the development of NAFLD and regulation of hepatic lipid metabolism. Activation of AMPK has an important regulatory role on lipid metabolism (Kamikubo et al., 2016;Garcia et al., 2019). Activated AMPK promotes lipid catabolism and ATP production, reduces mitochondrial ATP consumption to maintain the steady state of cellular energy, and regulates the activity of lipid metabolism-related enzymes to regulate fatty acid synthesis and oxidation in the liver (Hardie, 2015;Garcia and Shaw, 2017). AMPK inhibits sterol-regulatory element binding proteins-1c (SREBP-1c) activity, inhibits lipid synthesis, and reduces lipid deposition in the liver . SREBP is an endoplasmic reticulum (ER) membrane-bound transcription factor that activates genes encoding fatty acid synthesis and regulating cholesterol (Horton et al., 2002). SREBP-1c, one of the isoforms of the SREBP family, is a key transcription factor with the function of enhancing the expression of genes related to hepatic adipogenesis (Irimia et al., 2017). It is mainly distributed in the liver and adipose tissue. Overexpression of SREBP-1c will cause lipid metabolism disorders. Activation of AMPK can downregulate SREBP-1c expression, control fatty acid oxidation (Samovski et al., 2015). TG and TC production in the liver, reduce lipid accumulation in hepatocytes, improve lipid metabolism disorders in the liver, and alleviate NAFLD (Tang et al., 2016).
Artemisia capillaris (AC) is a dried above-ground part of Artemisia capillaris Thunb. or Artemisia scoparia Waldst. et Kit. which is commonly used in traditional medicine for the treatment of clearing heat and gallbladder. It mainly contains coumarins, flavonoids, organic acids, volatile oils, terpenoids and other chemical components (Yuan et al., 2017). Recent studies have found that, in addition to its pharmacological effects such as choleretic and hepatoprotective, AC also has a variety of pharmacological activities such as antipyretic, antiinflammatory, analgesic, and regulating lipid metabolism (Hsueh et al., 2021)。Organic acids such as chlorogenic acid and caffeic acid contained in AC exhibited potential antiobesity effects in mice induced by high-fat diet (HFD) (Cho et al., 2010). In addition, Ethanol extract of Artemisia scoparia (Galluzzi et al., 2018) has a modulating effect on lipid metabolism. The addition of SCO to HFD significantly reduced the levels of circulating glycerol and free fatty acids, and in vitro experiments revealed that SCO attenuated the lipolytic effect in TNF-α-induced cultured adipocytes (Boudreau et al., 2018).
In the present study, we evaluated the effects of WAC on a high-fat diet-induced mouse model of NAFLD and an oleic acid-induced HepG2 cell model. Then we explored the mechanisms by which WAC improves lipid accumulation in vivo and in vitro.

Preparation of WAC
The air-dried AC used in this study, produced in Gansu Province, China, was purchased from Harbin Songshantang Pharmaceutical Company, China. AC was identified by a professor from the Institute of Chinese Veterinary Medicine, Northeastern Agricultural University. Weigh 100 g of dried AC, add 800 ml of distilled water, soak for 30 min at room temperature, decoct for 1 h, and collect the decoction solution. Then, 600 ml of distilled water was added and the decoction was decocted again for 1 h. The decoction was collected. The decoction solution collected twice was mixed, and the decoction solution was filtered, centrifuged to remove insoluble substances, and lyophilized at -50°C for 48 h to a powdered solid (Huang et al., 2022). The average yield of AC was 15.6% (W/W). The lyophilized product was stored at -20°C. For cellular experiments, WAC was dissolved in DMEM and filtered (0.22 μm).
Frontiers in Pharmacology frontiersin.org 02 2.2 Identification of WAC active ingredients by LC-MS WAC (100 mg) were individually grounded with liquid nitrogen and the homogenate was resuspended with prechilled 80% methanol by well vortex. The samples were incubated on ice for 5 min and then were centrifuged at 15,000 g, 4°C for 20 min. Some of supernatant was diluted to final concentration containing 53% methanol by LC-MS grade water. The samples were subsequently transferred to a fresh Eppendorf tube and then were centrifuged at 15000 g, 4°C for 20 min (Want et al., 2013). Finally, the supernatant was injected into the LC-MS/MS system analysis LC-MS/MS analyses were performed using an ExionLCTM AD system (SCIEX) coupled with a QTRAP ® 6,500 + mass spectrometer (SCIEX). Samples were injected onto a Xselect HSS T3 (2.1 × 150 mm, 2.5 μm) using a 20-min linear gradient at a flow rate of 0.4 ml/min for the positive/negative polarity mode. The eluents were eluent A (0.1% Formic acid-water) and eluent B (0.1%Formic acid-acetonitrile) (Luo et al., 2015). The solvent gradient was set as follows: 2% B, 2 min; 2-100% B, 15.0 min; 100% B, 17.0 min; 100-2% B, 17.1 min;2% B, 20 min. QTRAP ® 6,500 + mass spectrometer was operated in positive polarity mode with Curtain Gas of 35 psi, Collision Gas of Medium, Ion Spray Voltage of 5500V, Temperature of 550°C, Ion Source Gas of 1:60, Ion Source Gas of 2:60. QTRAP ® 6,500 + mass spectrometer was operated in negative polarity mode with Curtain Gas of 35 psi, Collision Gas of Medium, Ion Spray Voltage of -4500V, Temperature of 550°C, Ion Source Gas of 1:60, Ion Source Gas of 2:60.

Experimental grouping and sample collection
Five-week-old male C57BL/6 mice were purchased from Changsheng Biotechnology (Changsheng, Liaoning, China). Animal experimental design strictly followed the guidelines of the International Association for the Evaluation and Accreditation of Laboratory Animal Care and was licensed by the Research Animal Protection Committee of Northeast Agricultural University. Mice were housed at 25 ± 1°C and in humidity-controlled chambers with natural light and dark cycles. After a 1-week adaptation period, mice were randomly divided into two groups: normal diet (ND) group (n = 6); and high-fat diet (HFD) group (n = 12). The nutritional analysis values and energy supply ratios of the high-fat diet are shown in Tables 1, 2, respectively. Mice in the high-fat diet group at week 16 formed a model of NAFLD . Twelve mice in the high-fat diet group were randomly divided into two groups: the NAFLD model group (n = 6), and the NAFLD + WAC group (HFD + WAC, 50 mg/kg/d, WAC gavage twice a week, n = 6). WAC gavage lasted for 6 weeks. The experimental period was 22 weeks. The doses administered in this experiment were selected based on the experiments of Choi (Choi et al., 2013). All mice were fed and watered ad libitum, and body weight and food intake were recorded weekly. At the end of the animal experimental period, all mice were fasted and watered ad libitum for 12 h. Then, blood was collected from the eyes after anesthesia with ether, and serum was obtained by centrifugation at 3,000 rpm for 5 min at 4°C. Livers were immediately excised, collected and stored at -80°C for analysis, or stored in 4% paraformaldehyde for histological studies.

Detection of major lipid indicators and glucose in blood and liver tissues
Serum TC, TG, Glucose, HDL and LDL levels were measured by a fully automated biochemical analyzer, and liver TC and TG levels were measured by a commercially available kit (Jiancheng, Nanjing, China). The liver tissue samples were accurately weighed, mechanically homogenized under ice and water bath conditions, centrifuged at 2,500 rpm for 10 min, and the supernatant was extracted and used to assay TG and TC levels.

Histological tests for liver pathology
Immediately after execution of the mice, liver tissue was fixed in 4% paraformaldehyde, paraffin-embedded, and sections were stained for H&E. The tissues were sectioned using a fcryostat and stained for Oil Red O. Light was observed using an optical microscope (Nikon, Tokyo, Japan).

Western blot analysis
Total protein samples were prepared using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors and quantified by the BCA protein assay kit. Equal amounts of proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight region of the target protein indicated by the pre-stained Marker was cut off and transferred to PVDF membrane or NC membrane, and the skimmed milk powder was closed at room temperature for 2 hours, and the primary antibody was added at the end of closure and incubated in the refrigerator at 4°C overnight. The primary antibodies are shown in Table 4. Then incubation with secondary antibody IgG (1:10,000) and preparation of ECL luminescent solution (meilunbio, Dalian, China) was performed for strip exposure. The strips were exposed using a 5,200 Tanon imaging system (Tanon science and technology, Shanghai, China). The developed protein bands were further quantified by ImageJ software and then compared with the grayscale values of the internal reference gene β-tubulin to calculate the relative expression levels of the target proteins measured in each of the above experimental groups.

HepG2 cell culture and drug treatment
HepG2 cells were provided by Dr. Zhang, College of Food, Northeastern Agricultural University. HepG2 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C and 5% CO 2 atmosphere. In the experiments, cells were starved in DMEM without FBS for 12 h. Subsequently, cells were cultured with DMEM containing FBS (negative control), OA-BSA complex (0.5 mM) (Xie et al., 2016) alone for 24 h to develop steatosis, and OA (0.5 mM) and WAC (1.5 mg/ml) were co-treated for 24 h. Next, cells were collected for subsequent experimental analysis. OA-BSA complexes were prepared by first dissolving OA in NaOH at 75°C by saponification and then adding the saponified OA to the medium containing 5% BSA and heating at 55°C until the solution became clear and transparent, resulting in a final concentration of 0.5 mM OA-BSA complexes.

HepG2 cell viability assay
Cell viability was assessed by cck-8 assay. HepG2 cells were inoculated at a density of 5 × 104 per well in 96-well plates with 100 μL of medium per well. Six replicate wells were set up and HepG2 cells were pre-cultured in an incubator at 37°C for 24 h. Then OA was co-treated with WAC (0.5 mg/ml, 1 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml, 3 mg/ml) for 24 h. 10 μL of cck-8 working solution was added to each well, protected from light, and incubated for 0.5 h in an incubator at 37°C. The absorbance at 450 nm was measured by an enzyme marker, and the cell survival rate (%) was calculated relative to the control group.

HepG2 cell lipid droplet observation
Oil Red O staining was used to assess the level of intracellular lipid droplets. The cells were washed 3 times with PBS, fixed with 4% paraformaldehyde for 10 min, and washed 3 times with PBS. The appropriate amount of staining washing solution was added to cover cells for 20 s and then aspirated and stained with modified Oil Red O staining solution for 20 min, and then the modified Oil Red O staining solution was removed with staining washing solution and washed with PBS. Finally, stained HepG2 cells were observed with an E100 light microscope (Nikon, Tokyo, Japan). For BODIPY staining, HepG2 cells were first inoculated in 24-well plates and treated with OA and WAC when the density reached 60%-70% for 24 h. Cells were fixed with 4% paraformaldehyde PBS for 1 h at room temperature, then washed 3 times with PBS for 5 min each, stained with 4 μM BODIPY493/503 at 37°C for 30 min, washed with PBS for 3 times, re-stained with DAPI for 5 min, and washed with PBS for 3 times. After blocking with PBS, the cell plates were placed on an inverted fluorescence microscope and the fluorescence signal was acquired and quantified using Image-Pro Plus software (version 3.0, Media Cybernetics, Bethesda, MD, United States) and the same parameters. Commercially available kits (Nanjing Jiancheng Bioengineering Co., Ltd. Nanjing, China) were used to determine intracellular TC and TG levels and to assess the extent of lipid accumulation. TG and TC levels were measured according to the manufacturer's instructions, and protein concentrations were determined by the Enhanced BCA Protein Assay Kit. Then, lipid levels/protein concentrations were calculated.

Immunofluorescence assay
HepG2 cells were plated in 24-well plates and treated with OA and WAC when the density reached 60%-70% for 24 h. Cells were fixed with 4% paraformaldehyde, washed, permeabilized, and closed. Cells were incubated with SREBP-1c (1:200) primary antibody overnight at 4°C in the refrigerator. Secondary antibody goat anti-rabbit IgG HampL/HRP (1:200) was added to the cells for 1 h. The nuclei were stained with DAPI (BL105A, Biosharp, Hefei, China) for 5 min. After blocking with PBS, the cell plates were placed on an inverted fluorescence microscope and the fluorescence signal was acquired and quantified using Image-Pro Plus software (version 3.0, Media Cybernetics, Bethesda, MD, United States) and the same parameters.
2.12 qRT-PCR analysis 0.1 g of liver tissue was weighed, total RNA was extracted and RNA concentration was detected according to the kit. RNA was reverse transcribed into cDNA using the kit, primers were synthesized by Sangon Biotech (Sangon Biotech Co., Ltd. Shanghai, China), primer design is shown in Table 3 qRT-PCR was performed using SYBR Green in a Light Cycler@480 System (Roche, Switzerland). reaction conditions were: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 s, denaturation at 60°C for 1 min, 40 cycles; and finally melting reaction. The mRNA expression level was quantified using β-actin as the control. The calculation method used was 2 −ΔΔCt method.

Statistical analysis of data
Data were expressed as mean ± standard deviation, and statistical analysis was performed using SPSS software 22.0. Statistical differences in quantitative data between groups and

Antibodies and Reagents
Producer significance of nonparametric comparisons between groups were analyzed using one-way ANOVA and Tukey's test. Plots were performed using Origin 2019. p < 0.05 was considered statistically significant.

WAC alleviates high fat diet-induced obesity and hepatic steatosis in nonalcoholic fatty liver disease mice
To determine the effect of WAC on NAFLD in vivo, a mouse model of NAFLD was constructed by feeding mice with HFD for 16 weeks, and WAC was given by gavage at 50 mg/kg/d for the next 6 weeks (Figure 2A). In terms of histopathology, WAC treatment significantly improved fat accumulation in the liver of HFD-fed mice ( Figures 2B,I). Mice fed with HFD showed higher weight gain.
weight gain was reduced after WAC treatment ( Figures 2C-E). The liver index, inguinal adiposity index and epididymal adiposity index were significantly increased in HFD-fed mice, and the above indices were significantly reduced after 6 weeks of WAC treatment. HFD enhanced the adiposity index and WAC treatment reduced the adiposity index, suggesting that the weight loss was associated with a reduction in the amount of adiposity ( Figures 2F-H). These results suggest that WAC treatment alleviates obesity and hepatic steatosis in HFD-induced NAFLD mice.

WAC ameliorates lipid metabolism disorders in high fat diet-induced nonalcoholic fatty liver disease mice
Excessive accumulation of hepatic lipids is due to a dysregulation of lipid metabolic homeostasis, which ultimately leads to hepatic steatosis. Mice fed HFD exhibited significantly higher serum TG and TC levels compared to mice in the ND group, and WAC treatment restored serum triglyceride and cholesterol levels to normal levels ( Figures  3A,B). Elevated serum glucose in mice fed HFD was also reversed by WAC, restoring normal levels ( Figure 3C), suggesting that WAC may regulate glucose metabolism in mice. Compared with mice in the ND group, serum levels of HDL were reduced and LDL levels were increased in mice fed HFD, and both reduced HDL levels and increased LDL levels were reversed after 6 weeks of WAC treatment (Figures 3D,E). In addition, the liver of HFD-induced NAFLD mice exhibited elevated TG and TC levels, which were significantly reduced after WAC treatment ( Figures 3F,G). These results suggest that WAC treatment ameliorates metabolic disorders in HFDinduced NAFLD mice.

WAC ameliorates OA-induced lipid accumulation in HepG2 cells
The cytotoxic effect of WAC was determined by cck-8, and 1.5 mg/ml was selected as the WAC concentration for the following in vitro experiments ( Figure 4B). In vitro, lipid accumulation in HepG2 cells was induced with 0.5 mM OA. The effect of WAC on lipid accumulation in HepG2 cells was assayed by co-treatment of HepG2 cells with WAC and OA ( Figure 4A). Compared with the NC group, the TG content of HepG2 cells in the OA group was significantly higher, whereas the TG content was significantly lower after treatment of HepG2 cells with WAC ( Figure 4C). In contrast to the in vivo results, the OA-induced TC of HepG2 cells did not change, nor did WAC treatment cause changes in the TC of HepG2 cells ( Figure 4D). The results of cytosolic oil red O staining showed a significant increase in lipid droplets in HepG2 cells in the OA group compared to the NC group. There was a decrease in lipid droplets in HepG2 cells in the WAC group compared to the OA group ( Figure 4E). After HepG2 cells were incubated with OA for 24 h, lipid accumulation was detected by staining with BODIPY493/503 (a specific lipophilic probe), which showed that OA significantly promoted lipid accumulation in HepG2 cells, and the green fluorescence intensity of HepG2 cells in the WAC group was diminished ( Figure 4F). These results suggest that in vitro, WAC ameliorates OA-induced lipid accumulation in HepG2 cells.

WAC activates PI3K/AKT and AMPK in vivo
Total PI3K and AKT and their phosphorylation levels, AMPK and its phosphorylation levels were detected in vivo by western blotting. Mice in the HFD group had elevated PI3K and AKT protein levels, decreased phosphorylated PI3K and AKT protein levels, decreased p-PI3K/PI3K and p-AKT/AKT levels, and significantly increased AMPK and p-AMPK protein levels in the liver. Mice in the WAC group had decreased PI3K and AKT protein levels, increased phosphorylated PI3K and AKT protein levels, increased p-PI3K/PI3K and p-AKT/AKT levels, and increased p-AMPK/AMPK (Figures 5A,B). Liver SREBP-1c protein Frontiers in Pharmacology frontiersin.org levels were significantly elevated in mice in the HFD group, and WAC treatment significantly decreased the elevated SREBP-1c protein levels ( Figures 5C,D). The qRT-PCR results showed that WAC decreased the expression of the lipid synthesis genes FASN, ACC, SREBP-1c and SCD-1 caused by the high-fat diet ( Figure 5E). These results suggest that WAC activates PI3K/AKT and AMPK and decreases SREBP-1c expression. Frontiers in Pharmacology frontiersin.org 09

WAC activates PI3K/AKT and AMPK in vitro
In vitro, total PI3K and AKT and their phosphorylation levels, AMPK and p-AMPK protein levels were measured by protein blotting, and the trends were the same as in vivo. The phosphorylated PI3K and AKT protein levels were decreased, p-PI3K/PI3K levels were decreased, AMPK protein levels were significantly increased and p-AMPK protein levels were decreased in HepG2 cells of OA group. After WAC treatment of HepG2 cells, the above results were reversed ( Figures 6A-D). As with the results of in vivo experiments, SREBP-1c protein levels were significantly elevated in HepG2 cells in the OA group compared with the NC group, and WAC treatment reversed the elevated SREBP-1 protein levels ( Figures 6C,D). Immunofluorescence staining of OA and WAC-treated cells using SREBP-1c antibody showed that WAC significantly reduced SREBP-1c expression in HepG2 cells ( Figure 6E). WAC decreased OA-induced mRNA expression of the elevated expression of the lipid synthesis genes FASN, ACC, SREBP-1c and SCD-1 ( Figure 6F).

WAC reduces lipogenesis in HepG2 cells
We further verified whether SREBP-1c acts as a target of WAC to reduce lipid synthesis. We treated HepG2 cells with the SREBP-1c inhibitor Fatostatin (FATO) in vitro and performed subsequent experiments ( Figure 7A). After WAC treatment of HepG2 cells, TG was significantly reduced, but  Figures 7D,E). The expression of adipogenesis-related proteins SCD1, FAS and DGAT2 was also examined, and WAC reduced the protein   Figure 7G). With the SREBP-1c inhibitor FATO, the trend of action was the same as that of WAC. The above results suggest that WAC reduces lipogenesis in HepG2 cells and is able to regulate the SREBP-1c pathway.

Discussion
HFD-fed C57BL/6 mice for 16 weeks were successfully constructed as a model of NAFLD. WAC intervention reversed the abnormal levels of TG, TC, GLU, HDL, LDL in serum and TG and TC in liver of mice induced by high-fat diet, and the results of liver pathology tests indicated that WAC reduced the number of hepatic lipid droplets. The results of in vitro experiments were also similar, indicating that WAC has the effect of promoting lipid metabolism and reducing the accumulation of lipids in the liver. By using LC/MS technology, Zhao et al. detected nine Nine chlorogenic acid analogues of AC, including chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, 3,5-dicaffeoylquinic acid, 4,5dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, chlorogenic acid methyl ester, cryptochlorogenic acid methyl ester, neochlorogenic acid methyl ester (Zhao et al., 2014). The active components of WAC were identified by LC-MS. Recent studies have found that chlorogenic acid (CGA) inhibits adipocyte differentiation and triglyceride (TG) accumulation , and caffeic acid (CA) reduces gene expression related to fatty acid synthesis and improves hepatic steatosis (Kim et al., 2018), therefore, we hypothesize that the active substances of WAC that promote lipid metabolism are chlorogenic acid analogues.
PI3K/AKT signaling pathway plays an important role in the development of NAFLD (Huang et al., 2018;Santoleri and Titchenell, 2019). Insulin is recruited through insulin receptor substrate (IRS) and activates PI3K. Activated PI3K signals to its downstream target kinase AKT (Osorio-Fuentealba and Klip, 2015), which inhibits GSK3. GSK3 promotes the degradation of mature SREBP-1c (Cross et al., 1995), which mediates lipid metabolism (Lagathu et al., 2006). SREBP-1c is a major regulator of fatty acid synthesis in the liver (Osborne and Espenshade, 2022). It can control the amount of TG in vivo by regulating acetyl coenzyme A carboxylase 1 (ACC1), fatty acid synthase (FASN), stearoyl coenzyme A desaturase (SCD-1) and other enzymes or proteins related to fatty acid synthesis (Wang et al., 2015). In mouse liver and in vitro cultured HepG2 cells, we observed that HFD and OA impaired PI3K/AKT signaling pathway and up-regulated SREBP-1c protein expression, whereas WAC increased PI3K/AKT phosphorylation and decreased SREBP-1c expression. WAC decreased TG content and reduced lipid deposition in liver and HepG2 cells. Although there are no reports of WAC affecting

FIGURE 8
Schematic diagram of the mechanism by which WAC improves NAFLD. WAC activates PI3K/AKT pathway, activates AMPK to promote SREBP-1c expression, thereby reducing hepatic TG synthesis and lipid synthesis.
Frontiers in Pharmacology frontiersin.org NAFLD through the PI3K/AKT signaling pathway, it has been shown that chlorogenic acid prevents HFD-induced hepatic steatosis and may achieve this effect by modulating intestinal flora and increasing GLP-1 secretion (Jiang et al., 2022). AMPK is a member of the serine/threonine kinase family and has the role of SREBP-1c, a factor that regulates energy homeostasis (Desjardins and Steinberg, 2018). In recent years, the AMPK signaling pathway has been shown to be associated with the alleviation of metabolic disorders in many studies on the treatment of NAFLD. Activation of AMPK signaling pathway is a common feature of NAFLD treatment (Chen et al., 2019;Zhou et al., 2021;Chen et al., 2022;Kim et al., 2022;Liao et al., 2022;Zhang and Feng, 2022). Activated AMPK signaling pathway in the liver reduces NAFLD mainly by reducing lipid production in the liver and regulating fatty acid oxidation in the liver von Loeffelholz et al., 2021). AMPK is an upstream kinase of SREBP-1c (Zhao et al., 2022). It can inhibit the expression of genes related to lipid synthesis such as SREBP-1c (Kim et al., 2020), and reduce hepatic TG synthesis. In this experiments, p-AMPK/AMPK and SREBP-1c protein expression were detected by western blotting. The results showed that WAC reversed the trend of decreased p-AMPK/AMPK protein expression and increased SREBP-1c expression in HFD mouse hepatocytes and OA-induced HepG2 cells. This indicated that the AMPK/SREBP-1c pathway was activated by WAC. The mRNA expression of the lipid synthesis genes FASN, ACC, SREBP-1c and SCD-1 was also observed by qRT-PCR. This showed that WAC significantly reduced the expression of the above lipid synthesis genes. In summary, WAC reduces TG synthesis in liver and HepG2 cells by activating PI3K/AKT pathway and AMPK to decrease SREBP-1c expression and alleviate hepatic steatosis. SCD1, FAS and DGAT2 are all downstream factors of SREBP-1c. FAS and SCD1 are known to be involved in the ab initio synthesis, esterification and desaturation of fatty acids. DGAT2 catalyzes the final step of TG production and accelerates hepatic lipid synthesis (Liu et al., 2012;Shimano and Sato, 2017). We found that AMPK could regulate the expression of SREBP-1c. To verify whether SREBP-1c is the target of WAC action, we further inhibited SREBP-1c with inhibitors in vitro and found that the results after inhibition were consistent with the trend of WAC action results. The expression of SCD1, FAS, and DGAT2 were all inhibited after inhibition of SREBP-1c. The above results indicate that SREBP-1c is the pathway of WAC action, and WAC can regulate the SREBP-1c pathway and reduce lipogenesis in HepG2 cells.
Our experiments showed that WAC can activate PI3K/AKT pathway and AMPK and thus reduce hepatic TG synthesis, but there are some limitations. In this experiment, we did not investigate the specific relationship between PI3K/AKT pathway and AMPK each with SREBP-1c expression by further experiments. This is the focus of our follow-up study.
In summary, our in vivo and in vitro studies showed that WAC activates PI3K/AKT pathway, activates AMPK to promote SREBP-1c expression, thereby reducing hepatic TG synthesis and lipid synthesis (Figure 8). Therefore, AC could be used as a potential health herb for improving NAFLD and obesity.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
All experiments were conducted in accordance with the NEAU animal use and ethics policy. All animal experiments were approved by the Northeastern Agricultural University Experimental Animal Welfare and Ethics Committee (NEAUEC-20210219).


PVJ1 Is Not the First Tailed Temperate Phage Infecting Bacteria from Genus Psychrobacillus. Comment on Liu et al. Isolation and Characterization of the First Temperate Virus Infecting Psychrobacillus from Marine Sediments. Viruses 2022, 14, 108

In a recent study published in Viruses [...].

In a recent study published in Viruses [1], the authors claim to have isolated and characterized the first tailed temperate phage able to infect a bacterium from the genus Psychrobacillus-Psychrobacillus phage PVJ1 (GenBank accession number: MZ983385). These claims, however, are misleading, as at least two tailed temperate siphophages infecting a Psychrobacillus sp. had their complete annotated genomes deposited in public biological sequence repositories prior to the public availability of phage PVJ1.
Based on the respective GenBank submission associated with the aforementioned paper, the complete annotated genome of Psychrobacillus phage PVJ1 (MZ983385.1) was deposited to GenBank on 31 August 2021 and subsequently released on 22 September 2021, while the complete annotated genomes of Psychrobacillus phages Perkons (MT325768.1) and Spoks (MT410774.1) were deposited to GenBank on 10 April 2020 and 29 April 2020, and were publicly released on 6 May 2020 and 27 May 2020, respectively, more than a year prior to phage PVJ1.
Although the authors performed a great job at describing the isolation and characterization of Psychrobacillus phage PVJ1, we found it quite disturbing that they, apparently, have not attempted a quick public biological sequence repository search using a query as simple as "Psychrobacillus phage" prior to claiming to have reported the first known phage infecting a member of the genus Psychrobacillus. This has prompted us to highlight the necessity of having a look into the International Nucleotide Sequence Database Collaboration (INSDC) databases (e.g., GenBank, European Nucleotide Archive, DNA Data Bank of Japan) before making any claims regarding newly isolated and characterized phages' place within the context of the known phage diversity (such as claiming that the phage in question is the first known virus to infect the members of bacterial genus "X").
To our knowledge, phages Perkons and Spoks are the first phages capable of infecting a bacterium belonging to the genus Psychrobacillus that had their complete annotated genomes publicly available. These phages, along with their host-Psychrobacillus sp. L4-were retrieved in the Latvian Biomedical Research and Study Centre from ice-free Antarctic soil material collected during the First Latvian Antarctic Expedition (K. Lamsters, J. Karuss, M. Krievans) that took place from 18 February to 4 April 2018 and received by us in the summer of the same year. Even though not formally published in the peer-reviewed literature yet, the process of isolation and characterization of these phages has been a major part of N. Zrelovs' MSc. thesis on the isolation and characterization of phages for bacterial genera, which had no known phages able to infect them yet, so as to expand the known cultured phage diversity, defended at the University of Latvia in summer 2020 [2]. The preliminary findings of these studies (including the presentation of the Viruses 2022, 14, 495. https://doi.org/10.3390/v14030495 https://www.mdpi.com/journal/viruses employed methodology, EM micrographs and annotation of a draft genome assembly for "Psychrobacillus virus L4F1" later to be refined and deposited to GenBank as "Psychrobacillus phage Perkons" under accession number MT325768.1) were also presented in the form of an oral presentation, "Isolation and characterization of novel bacteriophages from Antarctic soil samples", at the "Bioresources and Viruses IX" conference held in Kyiv, Ukraine (9-11 September 2019; presentation enclosed within the letter as Supplementary S1). The authors, apparently, have isolated and characterized the first known temperate myovirus capable of infecting a Psychrobacillus sp.-phage PVJ1-whose native host Psychrobacillus sp. GC2J1 came from marine sediments. However, overall, the phage PVJ1 still remains the third temperate tailed virus infecting a member of this bacterial genus following the temperate Psychrobacillus siphophages Perkons and Spoks, despite the fact the authors claim that " . . . there were no reports of bacteriophages infecting the genus Psychrobacillus . . . ", " . . . PVJ1 is the first bacteriophage isolated from Psychrobacillus" and " . . . PVJ1, the first-tailed temperate phage-infecting Psychrobacillus" within the paper [1]. Interestingly, although the host of phages Perkons and Spoks was initially classified by us based on 16S rRNA gene near-full-length sequencing as a strain of Psychrobacillus psychrodurans (on the basis of the closest 16S rRNA gene sequence match from cultured bacteria), the genus seems to have expanded shortly after with the addition of species that are more closely related to Psychrobacillus isolate L4-Psychrobacillus vulpis [3] and Psychrobacillus glaciei [4]-with the latter being isolated from an Antarctic iceberg and having the closest 16S rRNA gene sequence match to isolate L4 as of now ( Figure 1). ; 1000 replicates) was used to assess the node support. The tree is midpoint rooted and drawn to scale; branch lengths are in the unit of nucleotide substitutions per site. UFBoot support percentages are given for nodes having a support of more than 90%; these are also indicated by black rectangles. Tip labels are in the form of "Species/Strain/Accession".

Figure 1.
Maximum likelihood phylogeny of the partial 16S rRNA gene sequences of Psychrobacillus isolate L4 (host of phages Perkons and Spoks; tip label highlighted in green; sequence obtained using common 27F and 1492R primers), Psychrobacillus isolate GC2J1 (host of phage PVJ1; tip label highlighted in yellow) and validly named closely related bacterial isolates found in the EzBioCloud database [5]. Clustal-Omega v.1.2.4 [6] was used in auto mode for the multiple sequence alignment (MSA), which was then restricted to informative positions only using Gblocks [7] under default settings, resulting in the MSA of 40 sequences and 1250 positions (148 parsimony-informative, 87 singleton sites, 1015 constant sites). Maximum likelihood phylogeny reconstruction was performed using IQTREE v2.0.6. [8] under K2P+R2 as the best-fit substitution model according to ModelFinder [9] while allowing for polytomies. Ultrafast bootstrapping (UFBoot [10]; 1000 replicates) was used to assess the node support. The tree is midpoint rooted and drawn to scale; branch lengths are in the unit of nucleotide substitutions per site. UFBoot support percentages are given for nodes having a support of more than 90%; these are also indicated by black rectangles. Tip labels are in the form of "Species/Strain/Accession".
The growing accessibility of next-generation sequencing has remarkably impacted the rate of novel phage isolation and characterization studies conducted worldwide, resulting in a rapid expansion of the known phage diversity via steady growth of the complete phage genomes available in INSDC databases, albeit mostly with phages infecting a very limited number of different hosts. Although the peer-reviewed literature contains some attempts at summarizing the complete phage genome contents of the public databases (i.e., our attempt at doing so [11]), such studies, logically, provide no more than overview snapshots of the uncovered phage diversity at a given time and are always prone to becoming obsolete in the light of such diversity being ever-growing. To allow following the expansion of GenBank with novel phage entries, recently, a methodological framework that allows taking such "phage diversity snapshots" and even additionally extracting meaningful data for further phage comparative genomics analyses (INPHARED) was developed and published by Cook and colleagues [12] but requires some command line proficiency from the potential user seeking to run it independently of the (thus far monthly) output updates by the authors, making manual public database searches still a necessity for some researchers.
Despite the fact that there is a large fraction of complete annotated phage genome entries publicly available that still do not have an article or at least a genome announcement from the peer-reviewed literature linked to them for a variety of reasons (including Psychrobacillus phages Perkons and Spoks), we believe it is incorrect to ignore the existence of such entries when analyzing the place of any newly isolated phage within the context of known phages, regardless of the phages not being mentioned anywhere in the peerreviewed literature. With this letter, we would like to emphasize the self-evident need of phage researchers to make use of public biological sequence repositories to keep track of the currently available phage diversity, especially for supplementing claims requiring a high burden of proof (e.g., the isolated phage is the first known phage infecting "X", where "X" is a variable). As is exemplified by the case of the study describing the isolation and characterization of Psychrobacillus phage PVJ1, failure to do so might result in incorrect and misleading conclusions regarding the place of newly isolated phages within the thus far uncovered phage diversity, which is inevitably going to expand further beyond the currently recognized state, which still encompasses only a very limited variety of distinct hosts as of today.
Author Contributions: Conceptualization, methodology, formal analysis and investigation N.Z., A.D. and A.K.; resources and funding acquisition K.L., J.K. and M.K. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.


Identification of a Sialosides binding pocket in early-lineage SARS-CoV-2 via an optimized STD NMR method: a novel explanation for coronavirus virulence and zoonosis

the human cell surface and SARS-CoV-2 (“severe acute respiratory syndrome coronavirus 2) spike protein in Science an optimized saturation transfer difference (STD) NMR method that named as universal STA

Recently, Benjamin G. Davis group in Rosalind Franklin Institute and Oxford university revealed a novel interaction between sugars on the human cell surface and SARS-CoV-2 ("severe acute respiratory syndrome coronavirus 2) spike protein in Science by an optimized saturation transfer difference (STD) NMR method that named as universal STA [1].
It has been three years since Covid-19 became a global pandemic and it seems that SARS-CoV-2 will co-exist with human beings for a long time in future. Although it has been clearly demonstrated that SARS-CoV-2 enters human body via the classic binding between the spike protein receptor-binding domain (RBD) and human angiotensin converting enzyme-2 (ACE2) and the recognition mechanism was extensively studied [2], the interaction between SARS-CoV-2 spike protein and glycans on the human cell surface remains unclear. In this paper, the authors speculate that the N-term domain (NTD) of the spike protein in B-origin lineage SARS-CoV-2, which located adjacent to the RBD, can bind to Sialosides and thus influence the virulence and zoonosis of the virus. Besides, this binding is independent of the RBD-ACE2 interaction but may work cooperatively with it in the infection process (Fig. 1a).
Sialosides are a class of 9 carbon carboxylated monosaccharide derivatives, existing as short chain residues attached to the end of glycolipid and glycoprotein through α-2,3、α-2,6 or α-2,8 linkages. Sialosides play critical roles in viral and bacterial infections, for example, the interaction between hemagglutinin (HA) protein trimer on the surface of influenza viruses and Sialosides on the surface of host cell promotes viral attachment and consequent viral entry [3]. Furthermore, this binding is also observed in non-SARS coronaviruses including Middle East respiratory syndrome coronavirus (MERS-CoV), a close relative of SARS-CoV-2 [4]. The spike protein of SARS-CoV-2 has a structurally similar NTD with a putative Sialosides binding pocket [5], however, there is no clear clue that how its NTD works during infection process and the function of it remains confused. Charles et al. confirmed the interaction between spike NTD and Sialosides by using a new NMR method. They modified existing STD NMR through theoretical analysis and computational approach and overcome the rigorous constraint on binding rates for protein-ligand interaction, hence expanding its application. This altered method was named as uSTA due to the improved compatibility. Combined uSTA and Cryo-EM results demonstrated that spike NTD harbours a binding site for Sialosides that formed by H69, Y145, W152, S247 and Q183, which located on the outer surface of spike protein (Fig. 1b,  c). Sialosides binds to SARS-CoV-2 spike in an 'end-on' manner, and interestingly, despite the high sequence similarity with MERS-CoV, the structural superposition revealed a 12 Å distance between two sialic acid binding

Open Access
Molecular Biomedicine *Correspondence: yangdu@cuhk.edu.cn pockets. Besides, the secondary structure elements used for binding are different: in MERS spike structure (RCSB 6NZK) sialic acid located at the edge of the central β-sheet, whereas in this newly identified SARS-CoV-2 spike structure it is bound at the centre of the β-sheet. However, this feature disappeared in the subsequent alpha, beta, delta and Omicron variants. Theamino acids around this pocket were mutated in the SARS-Cov-2 variants and therefore lose the ability to bind Sialosides on the lung cell surface, which may facilitate the release of virus from cells of the respiratory stystem and increase the infectivity as we observed in the epidemiologic studies.
Considering that Sialosides attach on cell surfaces as glycol-conjugates of glycolipid and glycoprotein, the authors assumed that glycosylation function in human body also impacts on SARS-CoV-2 infection. By analysing whole exome sequencing data derived from 533 patients infected with SARS-CoV-2, the authors identified two genes (LGALS3BP and B3GNT8) related to glycan among the top five which affect disease severity. Genetic variants of these two genes are related to a decrease in severe disease outcome. These two genes are associated with N-linked-polyLacNAc-chains, and therefore the authors indicate that SARS-CoV-2 utilizes N-linked-polyLacNAc-chains as an anchor point to attach on host cells. Taken together, genetic analysis of patients from Italy in 2020 revealed that patients with mutations in two specific genes related to the type of cell surface Sialosides exhibit a lower ratio of severe disease. Combined with the previous experiment data, it demonstrated that it is hard for the original virus to enter the deep inside of the lung in patient with altered Sialosides type, resulting in a less severe disease. These observations are consistent with the fact that the virulence is alleviated in new variants including alpha, beta, delta and Omicron.
Altogether, Charles et al. demonstrated the presence of a Sialosides-binding site in NTD of SARS-CoV-2 spike protein and delineate the mechanism underlying the interaction in early B-origin lineage virus. Nevertheless, the Sialosides-binding site is abolished during the spread of SARS-CoV-2. These observations implicate that the ability to bind Sialosides is required for viruses during the spread from intermediate host to human beings; however, this interaction is abrogated to facilitate the release from cell surface and enhance spread ability during pandemic. These observations shed light on the zoonosis, virulence and pandemic mechanism of SARS-CoV-2, and will definitely provide help for the long-term virus evolution study. The new uSTA NMR method developed by Charles et al. could also be utilized to investigate various host-pathogen interactions including influenza viruses, MERS-CoV and other infective microbes on a molecular level. Fig. 1 The molecular mechanism of SARS-CoV-2 spike protein NTD domain interacts with Sialosides on human cell surface. a Graphical representation showing host-pathogen interactions. b Cryo-EM structure of spike protein that binds Sialosides to via its NTD, which located at the outer surface of spike protein. Sialosides (yellow) are shown in dots and the spike trimer (green) are shown in surface. c A close look to the binding site for Sialosides. The pocket is formed by H69, Y145, W152, S247 and Q183 in the spike NTD. Hydrogen bonds are show in yellow dash lines


s2fieldsofstudy: Biology Medicine

text:
Height and nigral neuron density in Parkinson’s disease

Background The dopaminergic system modulates growth hormone secretion and previous results have suggested a link between short stature and an increased risk of Parkinson’s disease (PD). Methods In 36 Lewy body spectrum disease (LBD) cases (PD = 22) and 19 controls, nigral TH-positive neuron densities were measured postmortem from midbrain sections and corrected with the Abercrombie method. Body measurements were collected from autopsies or patient records. Our aim was to investigate the possible relationship between height and the density of neurons in the substantia nigra pars compacta (SNc). Results SNc neuron density (n/mm2) had an inverse association with height, (R2 = 0.317, p < 0.0001) in patients. The association was not explained by weight, age, sex, brain weight, medication, or disease motor severity. The association was also separately observed in patients with PD (n = 22), but not in subjects who died without diagnosed neurological diseases. Conclusions Individual adult height may be connected to nigral neuron numbers in patients with LBDs, including PD.

Introduction
Human adult height is determined by genetic factors, nutrition, socioeconomic status, age, and health, but data on the association between height and morbidity are heterogeneous. The strongest negative associations between height and mortality have been reported in respiratory and cardiovascular diseases, whereas an opposite relationship has been reported between height and the risk for cancer and musculoskeletal diseases [1]. A combined epidemiological and genetic evaluation of the possible association between height and diseases confirmed a link between height and the risk for 32 diseases but found no link with psychiatric and neurological diseases [1].
However, the mean height of individuals with Parkinson's disease (PD) has been reported to be lower than that of controls, although the difference was significant only in men [2]. The authors hypothesized that the lower stature of PD patients may be associated with dopaminergic connections with the hypothalamic-pituitary axis during early brain development [2]. A similar association has been seen with dementia risk [3]. Studies have also associated PD with higher risk of osteoporosis, and it has been hypothesized that PD may affect into skeletal homeostasis [4]. Stature decline in PD could thus also be caused by various postural deformities in PD, such as camptocormia, antecollis (dropped head) and scoliosis [5].
The loss of dopaminergic neurons in the Substantia nigra pars compacta (SNc) is a key neuropathological change in Lewy body diseases (LBDs), but the factors that influence the number of dopaminergic neurons in PD are unclear. There is a large variation in the number of nigral Open Access *Correspondence: laasaa@utu.fi 1 Clinical Neurosciences, Faculty of Medicine, University of Turku, Turku, Finland Full list of author information is available at the end of the article neurons in patients with PD, and symptom severity, disease duration or treatment factors only partially explain the interindividual differences in SNc. Therefore, given the previously reported association between height and PD, we aimed to investigate the relationship between dopaminergic neuron density and height postmortem in patients with PD and in individuals who died without diagnosed neurological diseases.

Subjects and clinical data
Thirty-six postmortem cases of patients with LBDs and 19 healthy controls were included in this study. The included patients were collected from 168 postmortem cases with neuropathologically confirmed Lewy body pathology (database previously described in [6]). The inclusion criteria were available height and weight information and autopsy data, and acceptable quality of nigral tissue samples for neuronal counting. The most common clinical diagnosis was PD (n = 22). Retrospectively the patients were categorized to three clinical phenotypes: parkinsonism predominant (n = 13), dementia (n = 11), and mixed (n = 12). Height and weight measurements were collected from autopsies (n = 28) conducted at Turku University Hospital, where the weight was measured with a floor scale and height with a measuring stick from heels to the top of the head. For eight cases, the measurements were not available and were retrieved from clinical records (median (range) time from recorded body measurements to death was 4.0 (0.9-10.2) years). Nineteen controls (individuals who died without known neurological diseases) were individuals who were examined by forensic pathologists at the Finnish Institute for Health and Welfare, as described [7].

Neuropathology
Database selection criteria and neuropathological methods have been described in detail previously [6]. Briefly, formalin-fixed and paraffin-embedded midbrain at the level of origin of the third cranial nerve was sectioned at 8 µm thick sections and stained with Luxol Fast Blue to evaluate borders of substantia nigra pars compacta (magnification 1.0) and with tyrosine hydroxylase (TH) immunohistochemistry for calculation of dopaminergic neurons. The sections were scanned with a Pannoramic P25 Flash slide scanner and analyzed with CaseViewer software version 2.3.0.99276 (3D HISTECH Ltd, Budapest, Hungary). TH-positive neurons were systematically manually calculated from the SNc (magnification 20.0). The cell count was corrected with the Abercrombie method using the diameter of TH-positive nuclei (magnification 63.0 or higher) as described [6]. The median (range) time from death to autopsy was 5 (2-8) days, and the median time from autopsy to neuropathological examination was 23 (12-73) days (missing data for 2 patients). Controls were analyzed using the same protocol. The median (range) time from death to autopsy was 3 (1)(2)(3)(4)(5)(6)(7)(8), and the median time from autopsy to neuropathological examination was 20 .

Statistics
SPSS Statistics 25 for Windows (IBM Corp, Armonk, NY, USA) was used for statistical analyses. Correlations between neuron density and clinical factors were evaluated with Pearson's correlation or Spearman's correlation as appropriate. Linear regression analysis by the enter method was performed within the correlating variables. Differences between groups were analysed with independent sample t-tests or chi-square tests as appropriate.
Only two-tailed tests were evaluated and p-values < 0.05 were considered significant. The normality of distribution was evaluated with histograms and the Shapiro-Wilk test.
When excluding the eight patients with body measurements from clinical records, the correlation with height remained significant (Pearson r = 0.584, p = 0.001). There were no sex differences in patient SNc neuron density (p = 0.25). The correlation between height and SNc neuron density also remained significant when patients with depression were excluded from the analysis and when sex and age were included as covariates in the linear regression model (r = 0.58, p = 0.004, β = 0.082, SD = 0.023). Quadratic correlation (r 2 = 0.351) demonstrated the association slightly better than linear correlation (r 2 = 0.317) (Fig. 1A).

Discussion
The results of the present study suggest a link between adult height and SNc neuron density in patients with LBDs. This association was seen in patients with PD, but not in controls. Short stature in LBD patients may be one of the factors that explains interindividual differences in nigral neuron numbers.
A previous study reported that a shorter young adult height increases the risk of PD decades later. The increase in risk was seen particularly in young males with short stature [2]. Our results further demonstrate that PD patients with low height at autopsy have lower numbers of dopaminergic neurons than taller patients, an effect that was not explained by differences in demographics or clinical characteristics of the disease. Although the two studies differ considerably in terms of methodology and perspective (premorbid risk increase vs. postmortem neuron counts), both studies point to a possible link between low height and greater risk for dopaminergic degeneration. Dopamine seems to modulate body size in C. elegans [9], and dopamine D2 receptors regulate body size through growth hormone secretion [10]. Importantly, 6-hydroxydopamine administration to rats at the perinatal stage not only causes a severe loss of striatal dopaminergic innervation but reduces the growth rate of the animals [11]. Progressed PD often leads to inadequate nutritional status, weight loss and immobility, which can cause depleted bone density [4]. Moreover, in mice 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine seems to cause bone loss, but treatment with levodopa further reduces bone mass suggesting specific dopamine dependent mechanism [12]. Though our results should be considered preliminary and need verification with larger samples, they support a theory of shared factors determining adult height and vulnerability to neuronal degeneration.
We observed the association between SNc neuronal density and height only in patients with Lewy body pathology, and quadratic correlation demonstrated a slightly stronger relationship than the linear correlation. We did not see a similar correlation in somewhat younger individuals who had died without known neurological diseases and showed no neuropathological signs of degenerative diseases. It is possible that the association between height and SNc neuron density is specific to  LBDs. It should also be noted that correlation coefficients between neuronal density and height were moderate and SNc neuron density is just one of many factors affecting adult height in patients with Lewy body spectrum diseases. It is also possible that SNc neurons do not directly reflect hypothalamic dopamine function as a decline in SNc may not be associated with a decline of dopaminergic neurons in the arcuate nucleus. The hypothalamic neurodegeneration has in fact been associated with the symptom severity of PD, endocrine and autonomic dysfunction and some non-motor symptoms of PD but not with the severity of SNc degeneration [13]. Nevertheless, further studies with larger samples of LBD patients and neurologically healthy individuals are needed, including hypothalamic sampling besides SNc. It is also of interest that even though weight loss and low BMI have been associated with PD severity [14], we were not able to find a relationship between BMI and SNc pathology. It is clear that multiple factors affect BMI in advanced PD, such as gastrointestinal symptoms, dysphagia and anhedonia associated with depression [15]. It is likely that these factors could overrun and mask the possible minor association between BMI and neuronal loss particularly in relatively small sample of patients. The timing of the body measurements may also obscure possible associations to neurodegeneration. Most measurements were performed at autopsy, and weight measurements postmortem do not necessarily reflect the weight during adult life due to weight loss at end stages of the disease before death. The error caused by the age-related stature decline [8] was nevertheless corrected in our model. A limitation of this study is the retrospective collection of clinical data from patient histories which led to some unavailable and missing data. Potential factors such as the age of symptom onset, genetic background and highest adult height should be considered for further studies. As a clinicopathological investigation, the present study is also limited by delays between clinical and neuropathological findings.
To summarize, our results point to a link between adult height and dopaminergic neuronal density in patients with PD. This finding is in line with a previous study focusing on the risk of PD and height. This could reflect the interaction between dopaminergic neurotransmission and growth hormone secretion in human development.